University of Kentucky
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Horticulture Faculty Patents
Horticulture
6-1-1999
Isolated Spinach Ribulose-1,5-Bisphosphate
Carboxylase/Oxygenase Large Subunit εNMethyltransferase and Method of Inactivating
Ribulose-1,5-Bisphosphatase Carboxylase/
Oxygenase Large Subunit εN-Methyltransferase
Activity
Robert L. Houtz
University of Kentucky, [email protected]
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Recommended Citation
Houtz, Robert L., "Isolated Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large Subunit εN-Methyltransferase and
Method of Inactivating Ribulose-1,5-Bisphosphatase Carboxylase/Oxygenase Large Subunit εN-Methyltransferase Activity" (1999).
Horticulture Faculty Patents. 4.
http://uknowledge.uky.edu/horticulture_patents/4
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US005908972A
United States Patent [19]
[11]
Patent Number:
5,908,972
Houtz
[45]
Date of Patent:
Jun. 1, 1999
Ying et al., Molecular Rationale for the Absence of Methy
lation at Lysyl Residue 14 in the Large Subunit of Spinach
[54] ISOLATED SPINACH RIBULOSE-1,5
BISPHOSPHATE CARBOXYLASE/
OXYGENASE LARGE SUBUNIT ‘N
Rubisco, Plant Physiology (Supplement), vol. 111, No. 2
METHYLTRANSFERASE AND METHOD OF
(1996).
Black et al., Light—regulated Methylation of Chloroplast
Proteins, The Journal of Biological Chemistry, vol. 262, No.
20, pp. 9803—9807, (1987).
Houtz et al., Affinity Puri?cation of Ribulose—1,5—Bispho
INACTIVATING RIBULOSE-1,5
BISPHOSPHATASE CARBOXYLASE/
OXYGENASE LARGE SUBUNIT ‘N
METHYLTRANSFERASE ACTIVITY
sphate Carboxylase/Oxygenase Large Subunit ‘N—Methyl—
[75] Inventor: Robert L. Houtz, Lexington, Ky.
Transferase, Supplement to Plant Physiology, Annual Meet
[73] Assignee: University of Kentucky Research
Houtz et al., Identi?cation and Speci?city Studies of Ribu
ing of Plant Physiologists (1992) (343).
Foundation, Lexington, Ky.
lose—1,5—Bisphosphate Carboxylase/Oxygenase Large Sub
unit e—N—Methyltransferase, Supplement to Plant Physiol
ogy, Annual Meeting of Plant Physiologists (1992) (344).
[21] Appl. No.: 08/687,916
[22] Filed:
Napoli et al., Introduction of a Chimeric Chalcone Synthase
Gene into Petunia Results in Reversible Co—Suppression of
Homologous Genes in trans, The Plant Cell, vol. 2, pp.
Jul. 29, 1996
Related US. Application Data
[63]
[51]
279—289 (1990).
Continuation-in-part of application No. 08/391,000, Feb. 21,
gene expression in transgenic tomatoes, Nature, vol. 334
Int. Cl.6 ........................... .. A01H 5/00; C12N 15/29;
(1988).
C12N 15/54; C12N 15/82
[52]
US. Cl. ................................. .. 800/205; 800/DIG. 18;
800/DIG. 19; 800/DIG. 23; 800/DIG. 26;
800/DIG. 40; 800/DIG. 41; 800/DIG. 42;
800/DIG. 43; 800/DIG. 44; 536/232; 536/23.6;
435/69.1; 435/70.1; 435/1723; 435/193;
435/320.1; 435/252.3
[58]
Smith et al., Antisense RNA inhibition of polygalacturonase
1995, Pat. NO. 5,723,752.
Field Of Search .......................... .. 800/205, DIG. 18,
800/DIG. 19, DIG. 33, DIG. 26, DIG. 4044;
536/232, 23.6; 435/69.1, 70.1, 172.3, 193_320.1
[56]
References Cited
PUBLICATIONS
Wang et al., “Af?nity Puri?cation of Ribulose—1,5bisphos
phate Carboxylase/Oxygenase Large Subunit 6N—Methyl
transferase”, Protein Expression and Puri?cation 6:528—536
(1995).
Klein et al., “Cloning and Developmental Expression of
PEA Ribulose—l,5—bisphosphate carboxylase/oxygenase
Large Subunit N—Methyltransferase”, Plant Molecular Biol
ogy 27:249—261 (1995).
Houtz et al., “Posttranslation Modi?cations in the Amino—
Terminal
Region
of
the
Large
Subunit
of
Niemi et al., Protein Methylation in Pea Chloroplasts, Plant
Physiol., V0. 93, pp. 1235—1240 (1990).
Houtz et al., Partial Puri?cation and Characterization of
Ribulose—1,5—bisphosphate Carboxylase/Oxygenase Large
Subunit ‘N—Methyltransferase, Plant Physiol., vol. 97, pp.
913—920 (1991).
Eckes et al., Isolation and characterization of a light—induc
ible, organ—speci?c gene from potato and analysis of its
expression after tagging and transfer into tobacco and potato
shoot, Mol Gen Genet vol. 205, pp. 14—22 (1986).
Klein et al., Cloning and developmental expression of the
sucrose—phosphate—synthase gene from spinach, Planta, vol.
190, pp. 498—510 (1993).
Houtz et al., Partial Amino Acid Sequence of Ribulose—l,
5—Bisphosphate Carboxylase/Oxygenase Large Subunit
‘N—Methyl—Transferase, Supplement to Plant Physiology,
vol. 102, No. 1 (1993) (448).
Klein et al., Cloning and Expression of the Rubisco Large
Subunit Methyl—Transferase Gene from Pea, Supplement to
Plant Physiology, vol. 105, No. 1 (1994) (438).
Ying et al., “Organization and characterization of the ribu
lose—1,5—bisphosphate carboxylase/oxygenase large subunit
‘N—methyltransferase gene in tobacco,” Plant Molecular
Biology 32:663—671 (1996).
Ribulose—l5—Bisphosphate Carboxylase/Oxygenase from
Primary Examiner—Robert A. Wax
Several Plant Species”, Plant Physiology, 98:1170—1174
Assistant Examiner—Tekchand Saidha
(1992).
Houtz et al., “Post—Translational Modi?cations in the Large
Attorney, Agent, or Firm—Burns, Doane, SWecker &
Mathis, L.L.P.
Subunit of Ribulose Bisphosphate Carboxylase/Oxyge
nase”, Proc. Natl. Acad. Sci, USA, 86:1855—1859 (Mar.
[57]
1989).
ABSTRACT
The gene sequence for ribulose-1,5-bisphosphate
Mulligan et al., “Reaction—intermediate Analogue Binding
by Ribulose Bisphosphate Carboxylase/Oxygenase Causes
Speci?c Changes in Proteolytic Sensitivity: The Amino—Ter
carboxylase/oxygenase (Rubisco) large subunit (LS)
minal Residue of the Large Subunit is Acetylated Proline,”
Proc. Natl. Acad. Sci., USA, 85:1855—1859 (Mar. 1989).
in the LS is disclosed. In addition, the full-length cDNA
clones for Rubisco LSMT are disclosed. Transgenic plants
and methods of producing same Which have the Rubisco
Ying et al., “Molecular Rationale for the Absence of Methy
lation at Lysyl Residue 14 in the Large Subunit of Spinach
6N-methyltransferase (protein methylase III or Rubisco
LSMT) from a plant Which has a des(methyl) lysyl residue
LSMT gene inserted into the DNA are also provided.
Rubisco”, Plant Physiology (Supplement), 111(2): (Jun. 1,
Further, methods of inactivating the enzymatic activity of
1996).
Rubisco LSMT are also disclosed.
Klein et al. (1995). Plant Molecular Biology 27: 249—261,
Jan. 1, 1995.
21 Claims, 15 Drawing Sheets
U.S. Patent
Jun. 1,1999
Sheet 1 0f 15
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Sheet 15 0f 15
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FIG. 7B
7500 -
6000 -
CpHr3o(A-tcmeinv“l1-sy)mg
4500 ‘
3000 '
1500 "
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P-55
P-55-174
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S-38
5,908,972
1
2
ISOLATED SPINACH RIBULOSE-1,5
BISPHOSPHATE CARBOXYLASE/
“Calmodulin N-methyltransferase,” J. Biol. Chem. 261(15)
:7060—7069 (1986)), histone-H1 (SarnoW et al., “A histone
OXYGENASE LARGE SUBUNIT ‘ N
H4-speci?c methyltransferase properties, speci?city and
METHYLTRANSFERASE AND METHOD OF
INACTIVATING RIBULOSE-1,5
effects on nucleosomal histones,” Biochim. Biophys. Acta
655:349—358 (1981); and Tuck et al., “TWo histone
BISPHOSPHATASE CARBOXYLASE/
OXYGENASE LARGE SUBUNIT 6N
Euglena gracilis, ” J. Biol. Chem. 260(11):7114—7121
METHYLTRANSFERASE ACTIVITY
(1985)), and ribosomal proteins (Chang et al., “Puri?cation
H1-speci?c protein-lysine N-methyltransferases from
and properties of a ribosomal protein methylase from
10
(1975); Lobet et al., “Partial puri?cation and characteriZa
tion of the speci?c protein-lysine N-methyltransferase of
RELATED APPLICATIONS
This application is continuation-in-part of US. patent
application Ser. No. 08/391,000, ?led on Feb. 21, 1995, now
US. Pat. No. 5,723,752, Which is hereby incorporated by
reference in its entirety.
15
tems remains obscure. Trimethyllysine can serve as a meta
20
While carboXyl methylation of bacterial membrane proteins
plays a major role in chemotaXis (Clarke, “Protein carboXyl
25
methyltransferases: TWo distinct classes of enZymes,” Ann.
Rev. Biochem. 54: 479—506 (1985)). Evidence suggests that
methylation of Lys-115 in calmodulin affects certain activi
ties including in vitro NAD kinase activation (Roberts et al.,
“Trimethyllysine and protein function,” J. Biol. Chem. 261
Grant No. DE-FG05-92ER26075, aWarded by the Depart
ment of Energy. The Government may have certain rights in
this invention.
BACKGROUND OF THE INVENTION
(4): 1491—1494 (1986)), and in vivo susceptibility to ubiq
uitination (Gregori et al., “Bacterially synthesiZed vertebrate
1. Field of the Invention
This invention relates to ribulose-1,5-bisphosphate
calmodulin is a speci?c substrate for ubiquitination,” J. Biol.
carboXylase/oXygenase (Rubisco) large subunit (LS)
‘N-methyltransferase (protein methylase III or Rubisco
LSMT). This enZyme catalyZes methylation of the e-amine
of lysine-14 in the large subunit of Rubisco. Many plant
species contain methylated Lys-14 in the LS of Rubisco but
some do not (i.e., a des(methyl) lysyl residue in the LS). In
addition, the present invention relates to a gene and full
length cDNA clones for Rubisco LSMT. The present inven
tion further relates to transgenic plants and methods of
Chem. 262(6):2562—2567 (1987); and Gregori et al., “Spe
30
Rubisco.
2. Description of the Related Art
Protein methylation is a Widespread and common post
ci?c recognition of calmodulin from Dictyostelium discoi
deum by the ATP ubiquitin-dependent degradative
pathWay,” J. Biol. Chem. 260(9):5232—5235 (1985); but see
also Ziegenhagen et al., “Multiple ubiquitination of calm
odulin results in one polyubiquitin chain linked to
35
calmodulin,” FEBS Lett. 271(1,2):71—75 (1990); and
Ziegenhagen et al., “Plant and fungus calmodulins are
polyubiquitinated at a single site in a Ca2+-dependent
producing same Which have the Rubisco LSMT gene
inserted into the DNA. This invention also relates to a four
amino acid insert (WVQQ) Which inactivates the enZymatic
activity of Rubisco LSMT and thereby accounts for the
subsequent absence of trimethyllysine-14 in the LS of
YL32, a yeast ribosomal protein,” Biochim. Biophy. Acta
997:224—231 (1989)). HoWever, the biological function of
post-translational protein methylation in all but a feW sys
bolic precursor to carnitine (Paik et al., “Carnitine biosyn
thesis via protein methylation,” TIBS 2: 159—162 (1977)),
ACKNOWLEDGEMENT OF GOVERNMENT
SUPPORT
This invention Was made With Government support under
Escherichia coli Q13,” Biochemistry 14(22):4994—4998
manner,” FEBS Lett. 273(1,2):253—256 (1990)). Con?icting
40
45
reports (Farooqui et al., “Effect of Methylation on the
Stability of Cytochrome c of Saccharomyces cerevisiae in
vivo,” J. Biol. Chem. 256(10):5041—5045 (1981); Frost et
al., “Cytochrome c methylation,” Protein methylation, Ch.
4, pp. 59—76 (1990); and Frost et al., “Effect of enZymatic
methylation of cytochrome c on its function and synthesis,”
Int. J. Biochem. 22(10):1069—1074 (1990); versus Cessay et
translational modi?cation catalyZed by several different pro
al., “The relationship betWeen the trimethylation of lysine 77
tein methyltransferases (Paik et al., “Protein methylation,”
and cytochrome c metabolism in Saccharomyces
cerevisiae, ” Int. J. Biochem. 26(5):721—734 (1994); Cessay
et al., “Further investigations regarding the role of trimeth
yllysine for cytochrome c uptake into mitochondria,” Int. J.
in Freedman et al. (eds), The Enzymology of Posttransla
tional Modi?cations of Proteins, vol. 2, pp. 187—228, Aca
demic Press, London (1985)). Proteins Which contain trim
ethyllysyl residues include cytochrome c (Cessay et al.,
“The relationship betWeen the trimethylation of lysine 77
and cytochrome c metabolism in Saccharomyces
cerevisiae, ” Int. J. Biochem. 26(5):721—734 (1994); Cessay
et al., “Further investigations regarding the role of trimeth
yllysine for cytochrome c uptake into mitochondria,” Int. J.
Biochem. 23(7,8): 761—768 (1991); DiMaria et al., “Cyto
chrome c speci?c methylase from Wheat germ,” Biochem
istry 21:1036—1044 (1982); Farooqui et al., “Effect of
Methylation on the Stability of Cytochrome c of Saccharo
myces cerevisiae in vivo,” J. Biol. Chem. 256(10)
:5041—5045 (1981); and Farooqui et al., “In vivo studies on
yeast cytochrome c methylation in relation to protein
Biochem. 23(7,8):761—768 (1991)) also implicate methyla
tion of Lys-77 in cytochrome c as having a role in protein
55
ological and biochemical function performed by methyla
tion of protein bound lysyl residues have been restricted to
site-directed mutational analysis of the methylation site in
60
the target protein (Ceesay et al., “The relationship betWeen
the trimethylation of lysine 77 and cytochrome c metabolism
in Saccharomyces cerevisiae, ” Int. J. Biochem. 26(5)
:721—734 (1994); Cessay et al., “Further investigations
regarding the role of trimethyllysine for cytochrome c
synthesis, “J. Biol. Chem. 255(10):4468—4473 (1980)),
calmodulin (Han et al., “Isolation and kinetic characteriZa
tion of the calmodulin methyltransferase from sheep brain,”
Biochemistry 32:13974—13980 (1993); and RoWe et al.,
stability, heme incorporation, and mitochondrial transport. A
major limitation to elucidating the biological role of lysine
methylation in eukaryotes has been the absence of a protein
methylase III gene. Hence, molecular studies of the physi
65
uptake into mitochondria,”Int. J. Biochem. 23(7,8):761—768
(1991); and Roberts et al., “Expression of a calmodulin
methylation mutant affects the groWth and development of
5,908,972
3
4
transgenic tobacco plants,” Proc. Nat. Acad. Sci. USA
89:8394—8398 (1992)). These studies have been inconclu
leucine-rich repeat motifs (Ying et al., “Organization and
characterization of the ribulose-1,5-bisphosphate
sive as to the exact biological role of methylation of the
carboxylase/oxygenase large subunit ‘N-methyltransferase
e-amine of protein bound lysyl residues.
gene in tobacco,” Plant Molecular Biology (In press)).
Rubisco LSMT has high speci?c speci?city, methylating
Ribulose-1,5-bisphosphate carboxylase-oxygenase
(Rubisco) catalyzes the reduction of atmospheric CO2 dur
only Rubisco and only lysyl residue 14 in the LS. Of many
ing photosynthesis. A great deal is knoWn about the quater
nary structure, catalytic mechanism, active site residues, in
vivo regulatory mechanisms, and gene expression for this
plant species examined several contain methylated Lys-14 in
the LS of Rubisco, such as pea and tobacco, but some do not,
such as spinach and alfalfa (Houtz et al., “Post-translational
modi?cations in the large subunit of ribulose bisphosphate
carboxylase/oxygenase,” Proc. Natl. Acad. Sci. USA
86:1855—1859 (1989); Houtz et al., “Posttranslational modi
?cations in the amino-terminal region of the large subunit of
ribulose-1,5-bisphosphate carboxylase/oxygenase from sev
abundant enzyme, see, for example, AndreWs et al.,
“Rubisco: Structure, Mechanisms, and Prospects for
Improvement, ” in Hatch et al. (eds), The Biochemistry of
Plants, vol. 10, pp. 131—218. Academic Press, NeW York
(1987); Dean et al., “Structure, evolution, and regulation of
rbcS genes in higher plants,” Annu. Rev. Plant. Physiol.
eral plant species,” PlantPhysiol. 98:1170—1174 (1992); and
Plant Mol. Biol. 40: 415—439 (1989); and Mullet, “Chloro
plast development and gene expression,” Annu. Rev. Plant.
unpublished data). There has been no explanation for the
Physiol. Plant Mol. Biol. 39: 475—502 (1988). Higher plant
methylated state (i.e., a des(methyl) lysyl residue in the LS).
Further, since some plant species, such as spinach, Wheat,
corn (maize) and lettuce do not contain methylated Lys-14
Rubisco is a hexadecameric protein composed of eight
chloroplast-encoded large subunits (referred to herein as
“LS”) and eight nuclear-encoded small subunits (referred to
herein as “SS”). Synthesis of the LS is accompanied by
post-translational processing of the N-terminal domain
(Houtz et al., “Post-translational modi?cations in the large
subunit of ribulose bisphosphate carboxylase/oxygenase,”
Proc. Natl. Acad. Sci. USA 86:1855—1859 (1989); and
existence of Lys-14 in the LS of Rubisco in a non
in the LS of Rubisco (Houtz et al. “Posttranslational modi
?cations in the amino-terminal region of the large subunit of
ribulose-1,5-bisphosphate carboxylase/oxygenase from sev
eral plant species,” PlantPhysiol. 98:1170—1174(1992); and
25
Mulligan et al., “Reaction-intermediate analogue binding by
ribulose bisphosphate carboxylase/oxygenase causes spe
SUMMARY OF THE INVENTION
ci?c changes in proteolytic sensitivity: The amino-terminal
residue of the large subunit is acetylated proline,” Proc.
In vieW of the state of the art as previously described,
there thus exists a need in the art for a better understanding
Natl.Acaa'. Sci. USA 85:1513—1517 (1988)). The N-terminal
of post-translational protein methylation in plants. More
Met-1 and Ser-2 are removed and Pro-3 acetylated.
speci?cally, a better understanding for the molecular basis
for the absence of trimethylation-14 in the LS of Rubisco
Additionally, the LS of Rubisco from tobacco, muskmelon,
pea, and several other species is post-translationally modi
from certain plant species.
?ed by trimethylation of the e-amine of Lys-14 (Houtz et al.,
It is thus an object of the present invention to provide a
Rubisco LSMT gene.
It is a further object of the present invention to provide the
DNA and amino acid sequence for a Rubisco LSMT
enzyme.
It is a still further object of the present invention to
“Posttranslational modi?cations in the amino-terminal
region of the large subunit of ribulose-1,5-bisphosphate
carboxylase/oxygenase from several plant species,” “Plant
Physiol. 98:1170—1174 (1992); Houtz et al., “Post
translational modi?cations in the large subunit of ribulose
bisphosphate carboxylase/oxygenase,” Proc. Natl. Acad.
Sci. USA 86:1855—1859 (1989)). The enzyme responsible
provide full-length cDNA clones for Rubisco LSMT.
for this latter modi?cation is a highly speci?c chloroplast
localized S-adenosylmethionine (AdoMet):protein (lys)
‘N-methyltransferase (protein methylase III, Rubisco
LSMT, EC 2.1.1.43) (Houtz et al., “Post-translational modi
?cations in the large subunit of ribulose bisphosphate
carboxylase/oxygenase,” Proc. Natl. Acad. Sci. USA
86:1855—1859 (1989)).
Rubisco LSMT has been affinity puri?ed ~8000-fold from
pea chloroplasts and identi?ed as a monomeric protein With
a molecular mass of ~57 kDa (Wang et al., “Affinity
Puri?cation of Ribulose-1,5-bisphosphate Carboxylase/
Oxygenase Large Subunit ‘N-Methyltransferase,” accepted
by Protein Expression and Puri?cation (1995)). Recently,
Rubisco LSMT cDNAs have been cloned and sequenced
from pea and tobacco (Klein et al., “Cloning and develop
mental expression of pea ribulose-1,5-bisphosphate
carboxylase/oxygenase large subunit N-methyltransferase,”
Plant Molecular Biol. 27:249—261 (1995); Ying et al.,
“Organization and characterization of the ribulose-1,5
unpublished data), it Was once assumed that these same plant
species did not possess the Rubisco LSMT gene.
In a ?rst aspect, the present invention relates to a Rubisco
LSMT gene Which exists in a higher plant With a des
45
(methyl) lysyl residue in the LS of Rubisco. A particularly
preferred higher plant includes the spinach plant.
In a second aspect, the present invention relates to the
DNA and amino acid sequence for a Rubisco LSMT
enzyme.
In a third aspect, the present invention relates to a
recombinant vector including the Rubisco LSMT gene
described above. The vector is suitable for transforming
higher plants.
In a fourth aspect, the present invention relates to an
isolated or recombinant Rubisco LSMT enzyme encoded by
the Rubisco LSMT gene described above.
In a ?fth aspect, the present invention relates to a recom
binant or transgenic plant transformed With the Rubisco
LSMT gene described above.
In a sixth aspect, the present invention relates to a method
bisphosphate carboxylase/oxygenase large subunit
of inactivating Rubisco LSMT activity Which comprises
‘N-methyltransferase gene in tobacco,” Plant Molecular
Biology (In press)). The deduced amino acid sequences of
tobacco Rubisco LSMT has 64.5% identity and 75.3%
similarity With the sequence of pea Rubisco LSMT, and both
proteins contain several copies of a conserved imperfect
inserting a 4 amino acid sequence (SEQ ID NO.:1) insert
(WVQQ) into Rubisco LSMT.
In a further aspect, the present invention relates to a
method for preventing or reducing Rubisco LSMT activity
in a photosynthesizing plant comprising transforming a
5,908,972
5
6
photosynthesiZing plant With a recombinant vector wherein
the vector comprises a Rubisco LSMT gene With the 12
nucleotide insert.
ScaI and EcoRI respectively, electrophoresed on a 0.7%
agarose gel and processed for DNA gel-blot analysis by
hybridiZation to the rbcMT-S cDNA probe labeled With
digoXigenin-UTP. A rbcMT-S cDNA clone in BlueScript II
KS(+) digested With EcoRI corresponding to one copy Was
With the foregoing and other objects, advantages and
features of the invention that Will become hereinafter
apparent, the nature of the invention may be more clearly
used for copy number reconstitution.
FIG. 6 shoWs eXpression of both S38 and S40 mRNA in
understood by reference to the following detailed descrip
spinach leaves. RNase protection assays using a 785-nt
antisense riboprobe designed to protect a 775-nt of the S40
tion of the preferred embodiments of the invention and to the
appended claims.
10
BRIEF DESCRIPTION OF THE DRAWINGS
mRNA from nt-455 to nt-1229, and a 306-nt and 457-nt of
the S38 mRNA from nt-455 to nt-760 and from nt-761 to
nt-1217 respectively, Were carried out. Lanes 1, 2, 3, 4 and
5 are 2.5, 5, 10, 20 and 20 pg of spinach leave total RNA.
After hybridiZation all but lane 5 Were digested With 1:100
FIG. 1A illustrates the genomic organiZation and restric
tion map of rbcMT-S. EXons are shoWn as heavy black bars,
introns as horiZontal lines, and the auXon is indicated by an 15
dilution of RNases. Lane 5 Was digested With a 1:50 dilution
arroW.
of RNases (Ambion).
FIG. 1B is a diagrammatic representation of the S38 and
S40 cDNAs With coding regions as heavy black bars,
untranslated regions as open bars and the auXon as a shaded
bar.
FIG. 1C shoWs Probe I, Which is a 1056-bp SfrI fragment
With the 12-bp auXon, and Probe II, Which is a riboprobe for
the RNAase protection assay Which results in only one
775-nt fragment protected by S40 mRNA, and tWo 306-nt
and 457-nt fragments protected by S38 mRNA.
FIG. 1D depicts the strategy for PCR cloning and joining
20
protein; lanes 3, 5, 7 and 9, insoluble protein.
FIG. 7B is a bar graph representing Rubisco LSMT
activity from the different constructs corresponding to the
25
lanes in FIG. 7A.
DETAILED DESCRIPTION OF THE
INVENTION
different cDNA fragments. The restriction enZymes labeled
With stars Were used to ligate corresponding fragments.
The present invention relates to a Rubisco LSMT gene, its
Abbreviations for restriction sites: B, BglI; E, EcoRI; S,
SacI; Sc, ScaI; Sf, SfrI; Sp, Spel and X, XbaI.
FIG. 7A is a Western blot analysis of S-40, S-38, P-55 and
P-55-174 mRNAs expressed in E. coli. Lane 1, standard
markers; lanes 2 and 3, S-40; lanes 4 and 5, P-55; lanes 6 and
7, P-55-174; lanes 8 and 9, S-38; lanes 2, 4, 6 and 8, soluble
30
DNA and amino acid sequence encoding therefor, cDNA
clones thereof, and a four amino acid sequence insert Which
FIGS. 2A, 2B, 2C, 2D, 2E and 2F shoW the nucleotide
sequence of the rbcMT-S and the corresponding deduced
inactivates the enZymatic activity of Rubisco LSMT.
amino acid sequences. Introns are printed in loWer case
letters and eXons in upper case letters. The putative start and
stop codons are underlined. The 12 nucleotides and corre
sponding 4 amino acids representing the auXon sequence are
residues in peptides are abbreviated as recommended by the
In the present application, naturally occurring amino acid
35
IUPAC OIUB Biochemical Nomenclature Commission as
40
folloWs: Phenylalanine is Phe or F; Leucine is Leu or L;
Isoleucine is Ile or I; Methionine is Met or M; Norleucine is
Nle; Valine is Val or V; Serine is Ser or S; Proline is Pro or
P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr
of Y; Histidine is His or H; Glutamine is Gln or Q;
indicated by bold italic letters. The deduced polypeptide for
the S38 cDNA is underneath the one for the S40 cDNA that
contains the auXon. The oligonucleotide primers for
sequencing, PCR and RACE, are indicated by arroWs above
Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid
the nucleotide sequence. The primers labeled With a star are
derived from the conserved regions of pea and tobacco
Rubisco LSMTs.
FIGS. 3A, 3B and 3C are a comparison of the deduced
amino acid sequences of S38, S40, With tobacco and pea
Rubisco LSMTs. Identical residues are indicated by vertical
lines and similar residues by colons. Gaps introduced to
maXimiZe alignment are indicated by dashes. Potential
N-glycosylation sites are shoWn in bold. Leucine-rich
repeat-like motifs are underlined. The four amino acid
is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or
C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is
45
50
by the formula H2NCHR1COOH, Wherein R1 is: (1) a loWer
sequence, WVQQ, deduced from the 12-nt auXon is shoWn
alkyl group; (2) a cycloalkyl group of from 3 to 7 carbon
atoms; (3) a heterocycle of from 3 to 7 carbon atoms and 1
to 2 heteroatoms selected from the group consisting of
in bold italic letters. The conserved peptide sequences, from
Which the primers are derived to clone the rbcMT-S, are
indicated by arroWs.
55
FIG. 4 (SEQ ID NOS.: 26—30) illustrates alternative
splicing of intron M of rbcMT-S mRNA. The top portion
shoWs the sequence of intron III and ?anking regions.
ShoWn beloW are the tWo types of mRNAs (S40 and S38)
produced by alternative splicing. When the second 3‘splice
FIG. 5 is an analysis of the spinach genomic DNA. An
aliquot of 20 pg of spinach genomic DNA Was digested With
oXygen, sulfur, and nitrogen; (4) an aromatic or arylalkyl
residue of from 6 to 15 carbon atoms optionally having from
1 to 3 substituents on the aromatic nucleus selected from the
group consisting of hydroXyl, loWer alkoXy, amino, and
carboXyl; (5) alkylene-Y Where alkylene is an alkylene
60
site is utiliZed, the 12-nt auXon is retained to produce S40
mRNA (center), Which encodes a 55.5 kD polypeptide. If the
?rst 3‘splice site is utiliZed, the auXon is absent and S38
mRNA is produced (bottom), Which encodes a 55.0 kD
polypeptide.
Gly or G, and X is any amino acid.
Synthetic or non-naturally occurring amino acids refer to
amino acids Which do not naturally occur in vivo but Which,
nevertheless, can be incorporated into the peptide structures
described herein. Preferred synthetic amino acids are the
D-amino acids of naturally occurring L-amino acids as Well
as non-naturally occurring D and L amino acids represented
group of from 1 to 7 carbon atoms and Y is selected from the
group consisting of hydroXy, amino, cycloalkyl of from 3 to
7 carbon atoms, heterocyclic of from 3 to 7 carbon atoms
and 1 to 2 heteroatoms selected from the group consisting of
65
oXygen, sulfur and nitrogen, and —C(O)R2 Where R2 is
selected from the group consisting of hydrogen, loWer alkyl,
loWer alkoXy, and —NR3R4 Where R3 and R4 are indepen
dently selected from the group consisting of hydrogen and
5,908,972
7
8
lower alkyl; (6) alkylene-S(O)nR5 where n is 1 or 2, and R5
LSMT are critical to the plant cell for viability. Furthermore,
is a lower alkyl or lower alkylene.
the exceptionally tight and speci?c nature of the interaction
between Rubisco LSMT and des(methyl) forms of Rubisco
creates the possibility for the development of novel synthetic
polypeptide herbicides, whose target is the in vivo interac
tion between Rubisco LSMT and Rubisco, whose speci?city
crosses a group of plant species related only by the presence
of Rubisco LSMT, and whose target protein has no homo
logue in the entire animal kingdom. Finally, this same
af?nity of Rubisco LSMT for des(methyl) forms of Rubisco
also creates the possibility for the site and protein speci?c
Particularly preferred synthetic amino acids include, by
way of example, the D-amino acids of naturally occurring
L-amino acids, L-1-napthylalanine, L-2-naphthylalanine,
L-cyclohexylalanine, L-2-amino isobutyric acid, the sulfox
ide and sulfone derivatives of methionine, and the lower
alkoxy derivatives of methionine. “Peptide mimetics” are
also encompassed by the present invention and include
peptides having one or more of the following modi?cations:
peptides wherein one or more of the peptidyl [—C(O)
delivery of compounds into the chloroplast and to Rubisco,
for the potential manipulation of Rubisco activity and/or
NH—] linkages (bonds) have been replaced by a non
peptidyl linkage such as carbamate linkage [—OC(O)N <],
phosphonate linkage, amidate linkage, sulfonamide linkage,
and secondary amine linkage or with an alkylated peptidyl
15
from spinach (Spinach oleracea) is a des(methyl) form of
Rubisco with regards to trimethylation of lysyl residue 14 in
the large subunit (LS). In investigating the molecular basis
for the absence of trimethylation-14 in the LS of spinach
Rubisco, the inventor has isolated and sequenced two full
linkage [C(O)NR6— where R6 is a lower alkyl],
peptides wherein the N-terminus is derivatiZed to a
—NR7R8 group, to a —NC(O)R7 group where R7 and R8
are independently selected from hydrogen and lower alkyls
with the proviso that R7 and R8 are both not hydrogen, to a
length cDNAs (S40 and S38) and the gene for spinach
succinimide group, to a benZyloxycarbonyl-NH—(CBZ
NH—) group, to a benZyloxycarbonyl-NH— group having
from 1 to 3 substituents on the phenyl ring selected from the
group consisting of lower alkyl, lower alkoxy, chloro, and
bromo,
25
peptides wherein the C terminus is derivatiZed to >C(O)
R9 where R9 is selected from the group consisting of
Rubisco LSMT (rbcMT-S). This discovery was quite unex
pected since it was once thought that spinach did not possess
the Rubisco LSMT gene because it contained a des(methyl)
lysyl residue in the LS of Rubisco. The gene for spinach
Rubisco LSMT, covering all 6 exons and 5 introns, has an
organiZation similar to the tobacco Rubisco LSMT gene
(rbcMT-T). Southern blot analysis of spinach genomic DNA
hydrogen, lower alkyl, lower alkoxy, and NRloR11 where
shows that the rbcMT-S is present as a single copy. The
deduced amino acid sequence from the rbcMT-S cDNAs
R10 and R11 are independently selected from the group
consisting of hydrogen and lower alkyl.
shows 60% and 62% identity with the amino acid sequences
of pea and tobacco Rubisco LSMT, respectively.
Moreover, the particular sequence disclosed herein for the
spinach Rubisco LSMT gene may be used to determine the
Other abbreviations are as follows: aa, amino acid(s);
auxon, auxiliary exon; bp, base pair(s); nt, nucleotide(s);
Rubisco LSMT, Ribulose-1,S-bisphosphate carboxylase/
oxygenase large subunit ‘N-methyltransferase; RACE, rapid
stability.
Ribulose bisphosphate carboxylase/oxygenase (Rubisco)
35
ampli?cation of cDNA ends; RT-PCR, reverse transcription
polymerase chain reaction
Although the present invention is described with respect
to spinach, it will be appreciated that the techniques
employed herein are applicable to other plants species which
particular sequence in other photosynthesiZing plants. The
sequence of the gene may be used as a probe to screen cDNA
or genomic DNA libraries from other plants and, due to the
expected homology between the gene sequences in the
various plant species, the particular sequence for the
Rubisco LSMT gene in other species may then be found.
contain a des(methyl) form of Rubisco with regards to
In a further aspect, the present invention relates to a
trimethylation of lysyl residue 14 in the large subunit (LS).
Examples of such plant species include alfalfa, wheat, corn
(maiZe) and lettuce.
recombinant or transgenic plant transformed with the
Rubisco LSMT gene described above. The methods
Ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) large subunit (LS) ‘N-methyltransferase (referred
employed for transforming the plants are generally known in
45
the art. For example, the transformation method described in
Bechtold et al, PlantaAgrobacterium Mediated Gene Trans
to herein as “Rubisco LSMT”) catalyZes methylation of the
e-amine of lysine-14 in the LS of Rubisco. Rubisco is the
fer By In?ltration of Adult Arabidopsis Thaliana Plants,
world’s most abundant protein, and serves as the only
et al, “Agrobacterium tumefaciens-mediated transformation
of Arabidopsis thaliana root explants by using kanamycin
signi?cant link between the inorganic and organic carbon
pools in the Earth’s biosphere by catalyZing the reduction of
atmospheric carbon dioxide to carbohydrates during photo
synthesis. Perturbations of Rubisco activity translate directly
into similar changes in plant growth and yield. Thus, there
is signi?cant interest in the art in the potential manipulation
and control of Rubisco activity through genetic engineering.
CR. Acad. Sci., Paris 316:1194—1199 (1993) and Valvekens
55
selection,” Proc. Natl. Acad. Sci. USA 85:5536—5540
(1988), may be used in the method of the present invention.
To achieve the present invention, a full-length cDNA
clone was isolated by the present inventor utiliZing poly
merase chain reaction (PCR)-based technology and conven
tional bacteriophage library screening. PCR techniques are
disclosed, for example, in Klein et al, “Cloning and Devel
However, the complexity and multimeric nature of
Rubisco have proven to be substantial obstacles to achieving
this goal, which have not yet been overcome. Rubisco
opmental Expression of the Sucrose-Phosphate-Synthase
LSMT provides an opportunity for the selective manipula
tion of Rubisco activity through changes in the structure and
Ampli-Taq PCR kit by Perkin Elmer-Cetus, Emeryville,
stability of the N-terminal region in the LS, an area known
to be essential for catalytic activity. Rubisco LSMT is a
teriophage library screening is described, for example, in
Gene From Spinach,” Planta 190:498—510 (1993); in
Calif.); and in the manufacturer’s instruction manual. Bac
highly speci?c enZyme which is found to interact only with
Rubisco and does not interact with any other protein in the
plant cell. Since Rubisco catalyZes the reduction of atmo
spheric CO2 during photosynthesis, Rubisco and Rubisco
65
Gantt et al, “Transfer of rpl22 to the Nucleus Greatly
Preceded its loss from the Chloroplast and Involved the Gain
of an Intron,” EMBO J. 10:3073—3078 (1991), and in the
information provided by the manufacturer of the screening
membrane (Stratagene, La Jolla, Calif.).